RESUMEN
Although approximately 70% of bladder cancers are noninvasive and have high recurrence rates, early-stage disease is understudied. The lack of models to validate the contribution of molecular drivers of bladder tumorigenesis is a significant issue. Although mutations in PIK3CA are frequent in human bladder cancer, an in vivo model for understanding their contribution to bladder tumorigenesis is unavailable. Therefore, a Upk2-Cre/Pik3caH1047R mouse model expressing one or two R26-Pik3caH1047R alleles in a urothelium-specific manner was generated. Pik3caH1047R functionality was confirmed by quantifying Akt phosphorylation, and mice were characterized by assessing urothelial thickness, nuclear atypia, and expression of luminal and basal markers at 6 and 12 months of age. While at 6 months, Pik3caH1047R mice developed increased urothelial thickness and nuclear atypia, progressive disease was not observed at 12 months. Immunohistochemistry showed urothelium maintained luminal differentiation characterized by high forkhead box A1 (Foxa1) and peroxisome proliferator-activated receptor γ expression. Surprisingly, Pik3caH1047R mice subjected to low-dose carcinogen exposure [N-butyl-N-(4-hydroxybutyl)nitrosamine] exhibited no significant differences after exposure relative to mice without exposure. Furthermore, single-sample gene set enrichment analysis of invasive human tumors showed those with mutant PIK3CA did not exhibit significantly increased phosphatidylinositol 3-kinase/AKT pathway activity compared with wild-type PIK3CA tumors. Overall, these data suggest that Pik3caH1047R can elicit early tumorigenic changes in the urothelium, but progression to invasion may require additional genetic alterations.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt , Neoplasias de la Vejiga Urinaria , Animales , Humanos , Ratones , Carcinogénesis/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Mutación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Urotelio/metabolismoRESUMEN
UPII-mutant Ha-ras transgenic mice develop urothelial hyperplasia and low-grade papillary carcinoma, which mimics human non-muscle invasive bladder cancer (NMIBC). We investigated the effects and mechanisms of kawain, a main kavalactone in the kava plant, on oncogenic Ha-ras-driven urothelial carcinoma in these mice. The mice were fed at six weeks of age with vehicle control or kawain (6 g/kg) formulated food for approximately five months. Seventy-eight percent of the mice or more fed with kawain food survived more than six months of age, whereas only 32% control food-fed male mice survived, (p = 0.0082). The mean wet bladder weights (a surrogate for tumor burden) of UPII-mutant Ha-ras transgenic mice with kawain diet was decreased by approximately 56% compared to those fed with the control diet (p = 0.035). The kawain diet also significantly reduced the occurrence of hydronephrosis and hematuria in UPII-mutant Ha-ras transgenic mice. Histological examination and immunohistochemistry analysis revealed that vehicle control-treated mice displayed more urothelial carcinoma and Ki67-positive cells in the bladder compared to kawain treated mice. Global metabolic profiling of bladder tumor samples from mice fed with kawain food showed significantly more enrichment of serotonin and less abundance of xylulose, prostaglandin A2, D2 and E2 compared to those from control diet-fed mice, suggesting decreased shunting of glucose to the pentose phosphate pathway (PPP) and reduced inflammation. In addition, kawain selectively inhibited the growth of human bladder cancer cell lines with a significant suppression of 4E-BP1 expression and rpS6 phosphorylation. These observations indicate a potential impact of kawain consumption on bladder cancer prevention by rewiring the metabolic programs of the tumor cells.
Asunto(s)
Carcinoma de Células Transicionales , Neoplasias de la Vejiga Urinaria , Animales , Ratones , Transformación Celular Neoplásica , Ratones Transgénicos , Serina-Treonina Quinasas TOR , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. Although electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We have previously demonstrated that systemic Tamm-Horsfall protein (THP; uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we showed that the kidney increases circulating THP by enhancing the basolateral release of THP from medullary thick ascending limb cells. In patients with sepsis, changes in circulating THP were positively associated with a critical illness. THP was also found de novo in injured lungs. Genetic ablation of THP in mice led to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo led macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis, and chemotaxis, and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improved survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function, and systemic THP has therapeutic potential in sepsis.NEW & NOTEWORTHY Specific therapies to improve outcomes in sepsis with kidney injury have been limited by an unclear understanding of how kidney injury increases sepsis mortality. Here, we identified Tamm-Horsfall protein, known to protect in ischemic acute kidney injury, as protective in preclinical sepsis models. Tamm-Horsfall protein also increased in clinical sepsis without severe kidney injury and concentrated in injured organs. Further study could lead to novel sepsis therapeutics.
Asunto(s)
Lesión Renal Aguda , Sepsis , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Animales , Modelos Animales de Enfermedad , Riñón/metabolismo , Sepsis/complicaciones , Sepsis/metabolismo , Uromodulina/genética , Uromodulina/metabolismoRESUMEN
Rational selection of metal ions and organic ligands to synthesize metal-organic complexes (MOCs) is necessary for constructing multifunctional materials. Herein, we have obtained a novel heterotrimetallic Zn2Dy2Ir pentanuclear MOC by the assembly of DyIII, luminescent ZnII(valpn), and [IrIII(H2L)(ppy)2]Cl metalloligands (Hppy = 2-phenylpyridine, H2L = 2,2'-bipyridine-5,5'-di-p-benzoic acid). Single-crystal structural analysis shows that the central [IrIII(L)(ppy)2]- bridges two ZnDy moieties using two carboxylates of L2-. Measurements of organic light-emitting diodes (OLEDs) show that the maximum luminance is 284.2 cd/m2 and the turn-on voltage is 6 V. Magnetic studies reveal that Zn2Dy2Ir is a field-induced single-molecule magnet (SMM) with an energy barrier of 19.1(2) K under a 2 kOe dc field. Zn2Dy2Ir shows luminescence sensing with a quenching efficiency of up to 99.0% for 2,4,6-trinitrophenol (TNP).
RESUMEN
Integrating magnetic and optical properties into a metal-organic framework (MOF) remains a great challenge. Herein, we have reasonably constructed two 3D magnetooptical MOFs by incorporating a [IrIII(ppy)2(bpy)]+-based fluorescent metalloligand and magnetic LnIII centers. The alternating arrangements of Δ- or Λ-[IrIII(ppy)2(bpy)]+ endow these MOFs with enhanced optical properties. Moreover, the use of DyIII leads to field-induced slow magnetic relaxation. This work provides an effective strategy for the preparation of magnetooptical bifunctional MOFs.
RESUMEN
Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen.Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.
Asunto(s)
Infecciones Neumocócicas/prevención & control , Streptococcus pneumoniae , Células Th17/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/fisiología , Infecciones Neumocócicas/etiología , Prevotella melaninogenica , Streptococcus mitis , VeillonellaRESUMEN
Electronic-cigarettes (E-cigs) are marketed as a safe alternative to tobacco to deliver the stimulant nicotine, and their use is gaining in popularity, particularly among the younger population. We recently showed that mice exposed to short-term (12 wk) E-cig smoke (ECS) sustained extensive DNA damage in lungs, heart, and bladder mucosa and diminished DNA repair in lungs. Nicotine and its nitrosation product, nicotine-derived nitrosamine ketone, cause the same deleterious effects in human lung epithelial and bladder urothelial cells. These findings raise the possibility that ECS is a lung and bladder carcinogen in addition to nicotine. Given the fact that E-cig use has become popular in the past decade, epidemiological data on the relationship between ECS and human cancer may not be known for a decade to come. In this study, the carcinogenicity of ECS was tested in mice. We found that mice exposed to ECS for 54 wk developed lung adenocarcinomas (9 of 40 mice, 22.5%) and bladder urothelial hyperplasia (23 of 40 mice, 57.5%). These lesions were extremely rare in mice exposed to vehicle control or filtered air. Current observations that ECS induces lung adenocarcinomas and bladder urothelial hyperplasia, combined with our previous findings that ECS induces DNA damage in the lungs and bladder and inhibits DNA repair in lung tissues, implicate ECS as a lung and potential bladder carcinogen in mice. While it is well established that tobacco smoke poses a huge threat to human health, whether ECS poses any threat to humans is not yet known and warrants careful investigation.
Asunto(s)
Adenocarcinoma del Pulmón/inducido químicamente , Sistemas Electrónicos de Liberación de Nicotina , Hiperplasia/inducido químicamente , Neoplasias Pulmonares/inducido químicamente , Humo/efectos adversos , Fumar/efectos adversos , Adenocarcinoma del Pulmón/patología , Animales , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Hiperplasia/patología , Pulmón/patología , Neoplasias Pulmonares/patología , Masculino , Ratones , Nicotina/administración & dosificación , Vejiga Urinaria/patología , Urotelio/patologíaRESUMEN
E-cigarette smoke delivers stimulant nicotine as aerosol without tobacco or the burning process. It contains neither carcinogenic incomplete combustion byproducts nor tobacco nitrosamines, the nicotine nitrosation products. E-cigarettes are promoted as safe and have gained significant popularity. In this study, instead of detecting nitrosamines, we directly measured DNA damage induced by nitrosamines in different organs of E-cigarette smoke-exposed mice. We found mutagenic O6-methyldeoxyguanosines and γ-hydroxy-1,N2 -propano-deoxyguanosines in the lung, bladder, and heart. DNA-repair activity and repair proteins XPC and OGG1/2 are significantly reduced in the lung. We found that nicotine and its metabolite, nicotine-derived nitrosamine ketone, can induce the same effects and enhance mutational susceptibility and tumorigenic transformation of cultured human bronchial epithelial and urothelial cells. These results indicate that nicotine nitrosation occurs in vivo in mice and that E-cigarette smoke is carcinogenic to the murine lung and bladder and harmful to the murine heart. It is therefore possible that E-cigarette smoke may contribute to lung and bladder cancer, as well as heart disease, in humans.
Asunto(s)
Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Corazón/efectos de los fármacos , Pulmón/efectos de los fármacos , Nicotina/toxicidad , Nitrosaminas/toxicidad , Humo/efectos adversos , Vejiga Urinaria/efectos de los fármacos , Animales , Carcinogénesis/efectos de los fármacos , Línea Celular , Sistemas Electrónicos de Liberación de Nicotina , Humanos , Pulmón/metabolismo , Masculino , Ratones , Mutación/efectos de los fármacos , Nicotina/química , Nitrosaminas/química , Vejiga Urinaria/metabolismoRESUMEN
Tobacco smoke (TS) contains numerous cancer-causing agents, with polycyclic aromatic hydrocarbons (PAHs) and nitrosamines being most frequently cited as the major TS human cancer agents. Many lines of evidence seriously question this conclusion. To resolve this issue, we determined DNA adducts induced by the three major TS carcinogens: benzo(a)pyrene (BP), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanoe (NNK), and aldehydes in humans and mice. In mice, TS induces abundant aldehyde-induced γ-hydroxy-propano-deoxyguanosine (γ-OH-PdG) and α-methyl-γ-OH-PdG adducts in the lung and bladder, but not in the heart and liver. TS does not induce the BP- and NNK-DNA adducts in lung, heart, liver, and bladder. TS also reduces DNA repair activity and the abundance of repair proteins, XPC and OGG1/2, in lung tissues. These TS effects were greatly reduced by diet with polyphenols. We found that γ-OH-PdG and α-methyl-γ-OH-PdG are the major adducts formed in tobacco smokers' buccal cells as well as the normal lung tissues of tobacco-smoking lung cancer patients, but not in lung tissues of nonsmokers. However, the levels of BP- and NNK-DNA adducts are the same in lung tissues of smokers and nonsmokers. We found that while BP and NNK can induce BPDE-dG and O6-methyl-dG adducts in human lung and bladder epithelial cells, these inductions can be inhibited by acrolein. Acrolein also can reduce DNA repair activity and repair proteins. We propose a TS carcinogenesis paradigm. Aldehydes are major TS carcinogens exerting dominant effect: Aldehydes induce mutagenic PdG adducts, impair DNA repair functions, and inhibit many procarcinogens in TS from becoming DNA-damaging agents.
Asunto(s)
Aldehídos/toxicidad , Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Transformación Celular Neoplásica , Daño del ADN , Reparación del ADN/efectos de los fármacos , Neoplasias Pulmonares , Nitrosaminas/toxicidad , Contaminación por Humo de Tabaco/efectos adversos , Fumar Tabaco , Animales , Línea Celular , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Fumar Tabaco/efectos adversos , Fumar Tabaco/patologíaRESUMEN
X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptosis and plays key roles in the development, growth, migration, and invasion of cancer cells. Therefore, XIAP has recently attracted much attention as a potential antineoplastic therapeutic target, requiring elucidation of the molecular mechanisms underlying its biological activities. Here, using shRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, anchorage-independent growth assay, and invasive assay, we found that XIAP's RING domain, but not its BIR domain, is crucial for XIAP-mediated up-regulation of c-Myc protein expression in human bladder cancer (BC) cells. Mechanistically, we observed that the RING domain stabilizes c-Myc by inhibiting its phosphorylation at Thr-58 and that this inhibition is due to activated ERK1/2-mediated phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser-9. Functional studies further revealed that c-Myc protein promotes anchorage-independent growth and invasion stimulated by the XIAP RING domain in human BC cells. Collectively, the findings in our study uncover that the RING domain of XIAP supports c-Myc protein stability, providing insight into the molecular mechanism and role of c-Myc overexpression in cancer progression. Our observations support the notion of targeting XIAP's RING domain and c-Myc in cancer therapy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Neoplasias de la Vejiga Urinaria/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Invasividad Neoplásica/genética , Fosforilación/genética , Dominios Proteicos , Estabilidad Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Biology has taught us that a protein as abundantly made and conserved among species as Tamm-Horsfall protein (THP or uromodulin) cannot just be a waste product serving no particular purpose. However, for many researchers, THP is merely a nuisance during urine proteome profiling or exosome purification and for clinicians an enigmatic entity without clear disease implications. Thanks to recent human genetic and correlative studies and animal modeling, we now have a renewed appreciation of this highly prevalent protein in not only guarding urinary homeostasis, but also serving as a critical mediator in systemic inter-organ signaling. Beyond a mere barrier that lines the tubules, or a surrogate for nephron mass, mounting evidence suggests that THP is a multifunctional protein critical for modulating renal ion channel activity, salt/water balance, renal and systemic inflammatory response, intertubular communication, mineral crystallization and bacterial adhesion. Indeed, mutations in THP cause a group of inherited kidney diseases, and altered THP expression is associated with increased risks of urinary tract infection, kidney stone, hypertension, hyperuricemia and acute and chronic kidney diseases. Despite the recent surge of information surrounding THP's physiological functions and disease involvement, our knowledge remains incomplete regarding how THP is normally regulated by external and intrinsic factors, how precisely THP deficiency leads to urinary and systemic pathophysiology and in what clinical settings THP can be used as a theranostic biomarker and a target for modulation to improve patient outcomes.
Asunto(s)
Homeostasis , Hipertensión/fisiopatología , Hiperuricemia/fisiopatología , Cálculos Renales/fisiopatología , Mutación , Infecciones Urinarias/fisiopatología , Uromodulina/metabolismo , Animales , Biomarcadores/análisis , Humanos , Uromodulina/genéticaRESUMEN
Up to 15% of the population have mild to moderate chronic hypomagnesemia, which is associated with type 2 diabetes mellitus, hypertension, metabolic syndrome, and chronic kidney disease. The kidney is the key organ for magnesium homeostasis, but our understanding of renal magnesium regulation is very limited. Uromodulin (UMOD) is the most abundant urinary protein in humans, and here we report that UMOD has a role in renal magnesium homeostasis. Umod-knockout (Umod-/-) mice excreted more urinary magnesium than WT mice and displayed up-regulation of genes promoting magnesium absorption. The majority of magnesium is absorbed in the thick ascending limb. However, both mouse strains responded similarly to the diuretic agent furosemide, indicating appropriate function of the thick ascending limb in the Umod-/- mice. Magnesium absorption is fine-tuned in the distal convoluted tubule (DCT) via the apical magnesium channel transient receptor potential melastatin 6 (TRPM6). We observed decreased apical Trpm6 staining in the DCT of Umod-/- mice. Applying biotinylation assays and whole-cell patch-clamp recordings, we found that UMOD enhances TRPM6 cell-surface abundance and current density from the extracellular space. UMOD physically interacted with TRPM6 and thereby impaired dynamin-dependent TRPM6 endocytosis. WT mice fed a low-magnesium diet had an increased urinary UMOD secretion compared with the same mice on a regular diet. Our results suggest that increased urinary UMOD secretion in low-magnesium states reduces TRPM6 endocytosis and thereby up-regulates TRPM6 cell-surface abundance to defend against further urinary magnesium losses.
Asunto(s)
Homeostasis , Riñón/química , Magnesio/metabolismo , Canales Catiónicos TRPM/metabolismo , Uromodulina/fisiología , Animales , Endocitosis , Furosemida/farmacología , Humanos , Túbulos Renales Distales/metabolismo , Magnesio/orina , Ratones , Ratones Noqueados , Uromodulina/genéticaRESUMEN
Tamm-Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP-/- mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP-/- mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP-/- mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP-/- mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.
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Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/patología , Fagocitos/efectos de los fármacos , Fagocitos/fisiología , Uromodulina/genética , Uromodulina/metabolismo , Lesión Renal Aguda/etiología , Animales , Plasticidad de la Célula/genética , Proliferación Celular/efectos de los fármacos , Activación Enzimática , Humanos , Riñón/patología , Ratones , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Daño por Reperfusión/complicaciones , Uromodulina/química , Uromodulina/farmacología , Uromodulina/uso terapéuticoRESUMEN
Epigenetic aberrations are prominent in bladder cancer (BC) and contribute to disease pathogenesis. We characterized histone deacetylase (HDAC) expression, a family of deacetylation enzymes, in both in vitro and in vivo BC model systems and analyzed expression data from The Cancer Genome Atlas (TCGA). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis was used to determine the expression status of Class I and II HDACs in ten human BC cell lines, while qRT-PCR was used to determine HDAC expression in 24 human tumor specimens. The TCGA cohort consists of 408 muscle invasive BC (MIBC) clinical samples and analysis of this data set identified expression of HDAC4 and -9 as being associated with basal-squamous disease. These findings agree with qRT-PCR results identifying increased expression of HDAC4, -7, and -9 in basal BC cell lines (p < 0.05; Kruskal-Wallis test) and in clinical specimens with invasive bladder cancer (not statistically significant). We also observed increased expression in Hdac4, -7, and -9 in commonly used BC mouse models. Here, we identify suitable preclinical model systems for the study of HDACs, and show increased expression of Class IIa HDACs, specifically HDAC4 and HDAC9, in basal BC cell lines and in invasive clinical specimens. These results suggest this class of HDACs may be best suited for targeted inhibition in patients with basal BC.
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Histona Desacetilasas/genética , Neoplasias de la Vejiga Urinaria/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/metabolismo , Humanos , Ratones , Vejiga Urinaria/embriología , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Bladder cancer (BC) is the sixth most common cancer in the United States and is the number one cause of death among patients with urinary system malignancies. This makes the identification of invasive regulator(s)/effector(s) as the potential therapeutic targets for managing BC a high priority. p63 is a member of the p53 family of tumor suppressor genes/proteins, plays a role in the differentiation of epithelial tissues, and is believed to function as a tumor suppressor. However, it remains unclear whether and how p63 functions in BC cell invasion after tumorigenesis. Here, we show that p63α protein levels were much higher in mouse high-invasive BC tissues than in normal tissues. Our results also revealed that p63α is crucial for heat shock protein 70 (Hsp70) expression and subsequently increases the ability of BC invasion. Mechanistic experiments demonstrated that p63α can transcriptionally up-regulate Hsp70 expression, thereby promoting BC cell invasion via the Hsp70/Wasf3/Wave3/MMP-9 axis. We further show that E2F transcription factor 1 (E2F1) mediates p63α overexpression-induced Hsp70 transcription. We also found that p63α overexpression activates E2F1 transcription, which appears to be stimulated by p63α together with E2F1. Collectively, our results demonstrate that p63α is a positive regulator of BC cell invasion after tumorigenesis, providing significant insights into the biological function of p63α in BC and supporting the notion that p63α might be a potential target for invasive BC therapy.
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Factor de Transcripción E2F1/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/patología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Factor de Transcripción E2F1/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Masculino , Ratones , Invasividad Neoplásica , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Expression of Tamm-Horsfall protein (THP or uromodulin) is highly restricted to the kidney thick ascending limb (TAL) of loop of Henle. Despite the unique location and recent association of THP gene mutations with hereditary uromodulin-associated kidney disease and THP single nucleotide polymorphisms with chronic kidney disease and hypertension, the physiological function(s) of THP and its pathological involvement remain incompletely understood. By studying age-dependent changes of THP knockout (KO) mice, we show here that young KO mice had significant salt and water wasting but were partially responsive to furosemide, due to decreased luminal translocation of Na-K-Cl cotransporter 2 (NKCC2) in the TAL. Aged THP KO mice were, however, markedly oliguric and unresponsive to furosemide, and their NKCC2 was localized primarily in the cytoplasm as evidenced by lipid raft floatation assay, cell fractionation, and confocal and immunoelectron microscopy. These aged KO mice responded to metolazone and acetazolamide, known to target distal and proximal tubules, respectively. They also had marked upregulation of renin in juxtaglomerular apparatus and serum, and they were hypertensive. Finally, the aged THP KO mice had significant upregulation of Na-coupled urate transporters Slc5a8 and Slc22a12 as well as sodium-hydrogen exchanger 3 (NHE3) in the proximal tubule and elevated serum uric acid and allantoin. Collectively, our results suggest that THP deficiency can cause progressive disturbances in renal functions via initially NKCC2 dysfunction and later compensatory responses, resulting in prolonged activation of the renin-angiotensin-aldosterone axis and hyperuricemia.
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Hipertensión/metabolismo , Hiperuricemia/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , Micción , Uromodulina/deficiencia , Factores de Edad , Animales , Presión Sanguínea , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Modelos Animales de Enfermedad , Diuréticos/farmacología , Predisposición Genética a la Enfermedad , Hipertensión/genética , Hipertensión/fisiopatología , Hiperuricemia/genética , Hiperuricemia/fisiopatología , Riñón/fisiopatología , Riñón/ultraestructura , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/genética , Enfermedades Renales/fisiopatología , Masculino , Microdominios de Membrana/metabolismo , Ratones de la Cepa 129 , Ratones Noqueados , Transportadores de Ácidos Monocarboxílicos , Oliguria/genética , Oliguria/metabolismo , Oliguria/fisiopatología , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Fenotipo , Sistema Renina-Angiotensina , Intercambiador 3 de Sodio-Hidrógeno/genética , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Micción/efectos de los fármacos , Uromodulina/genéticaRESUMEN
Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIß was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIß expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIß expression and reduced cancer cell invasion, whereas RhoGDIß expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIß mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIß in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIß axis promoted BC invasion and lung metastasis.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/biosíntesis , Neoplasias Pulmonares/secundario , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Proteína Inhibidora de la Apoptosis Ligada a X/biosíntesis , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Animales , Línea Celular Tumoral , Femenino , Células HCT116 , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Invasividad Neoplásica , ARN Mensajero/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , NucleolinaRESUMEN
Bacterial access to the gut immune system is a crucial process to promote host immune responses. The probiotic L-92 strain of Lactobacillus acidophilus exerts anti-allergic immunomodulatory effects upon oral administration in mice. Here, we show that microfold cells (M cells) are responsible for L-92 internalization for evoking L-92-mediated immune responses. L-92 specifically bound to uromodulin, a glycosylphosphatidylinositol-anchored protein expressed exclusively on M cells among intestinal epithelial cells. Internalization of L-92 into M cells was significantly reduced in uromodulin-deficient (Umod-/-) mice compared to Umod+/+ mice. Furthermore, the binding of L-92 to uromodulin was significantly decreased after removal of surface layer protein A (SlpA) from the bacteria. Our study thus revealed a crucial role of uromodulin on the M-cell surface for the uptake of SlpA-positive lactic acid bacteria into M cells, possibly leading to subsequent delivery of the bacteria to dendritic cells closely associated with M cells for immunomodulation. Our study also shed light on the possibility that SlpA and uromodulin could be used as vehicle and target, respectively, for efficient mucosal vaccine delivery.
Asunto(s)
Proteínas Bacterianas/metabolismo , Células Dendríticas/inmunología , Mucosa Intestinal/metabolismo , Lactobacillus acidophilus/fisiología , Uromodulina/metabolismo , Animales , Células Cultivadas , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Probióticos , Unión Proteica , Transcriptoma , Uromodulina/genéticaRESUMEN
Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.
Asunto(s)
Adhesinas de Escherichia coli/metabolismo , Anticuerpos Monoclonales/inmunología , Adhesión Bacteriana , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Escherichia coli Uropatógena/metabolismo , Animales , Femenino , Masculino , Ratones Endogámicos C57BL , Modelos MolecularesRESUMEN
Cell migration is a dynamic process that is central to a variety of physiological functions as well as disease pathogenesis. The modulation of cell migration by p27 (officially known as CDKN1B) has been reported, but the exact mechanism(s) whereby p27 interacts with downstream effectors that control cell migration have not been elucidated. By systematically comparing p27(+/+) mouse embryonic fibroblasts (MEFs) with genetically ablated p27(-/-) MEFs using wound-healing, transwell and time-lapse microscopic analyses, we provide direct evidence that p27 inhibits both directional and random cell migration. Identical results were obtained with normal and cancer epithelial cells using complementary knockdown and overexpression approaches. Additional studies revealed that overexpression of manganese superoxide dismutase (MnSOD, officially known as SOD2) and reduced intracellular oxidation played a key role in increased cell migration in p27-deficient cells. Furthermore, we identified signal transducer and activator of transcription 3 (STAT3) as the transcription factor responsible for p27-regulated MnSOD expression, which was further mediated by ERK- and ATF1-dependent transactivation of the cAMP response element (CRE) within the Stat3 promoter. Collectively, our data strongly indicate that p27 plays a crucial negative role in cell migration by inhibiting MnSOD expression in a STAT3-dependent manner.